Biotherapeutic drugs, which may be defined as being derived from living organisms, and includes both proteins and DNA, comprise an important class of medicines that have attracted significant interest from both academic and commercial environments Examples of such drugs include monoclonal antibodies (mABs), Human growth hormone (hGH), insulin and glucagon. In the last decade in particular, a remarkable increase in the research, development and clinical use of biotherapeutic drugs for the treatment of a wide range of diseases, including various cancers, rheumatoid arthritis and multiple sclerosis, has been observed. Many of the currently approved mAB-based drugs (as approved by the US Food and Drug Administration) have been approved in the past five years.
Strict regulatory guidelines require that biotherapeutic drug products are clinically safe. This can significantly lengthen the time before a drug becomes available on the market. Product-specific requirements are, in part, satisfied through full characterisation of the molecular properties, solution behaviour and stability of the biotherapeutic drug formulation and, ultimately, by the demonstrable manufacture of a pure, homogeneous product.
One of the main challenges associated with the production of pure and homogenous biotherapeutic drugs is the formation of aggregates. An aggregate may be defined as an assembly of n monomers (where n>1), a monomer being defined as the most basic, non-divisible, unit of an intact protein molecule. Although natural aggregates, in the form of dimers, trimers and other higher order associations, can occur in some proteins and are essential for proper physiological function, for example in Thyroglobulins, Superoxide distumases and Aldolases; for mABs and other protein-based drugs the formation of aggregates is not required for drug function and instead can adversely affect their role as biotherapeutic agents. The immunogenic potency, i.e. the degree to which a molecule (biological or otherwise) is able to elicit an immunological response, of large protein aggregates that may be made up of several million protein molecules has been widely documented, for example via draft FDA guidance on immunogenicity assessment for therapeutic products issued in 2013 (available from www.fda.org). Whilst potency is a desired attribute for any therapeutic drug, the inherent heterogeneity of aggregate structures is problematic from manufacturing and quality control (QC) perspectives. Presently, the currently limited understanding of aggregate formation does not allow for the controlled and reproducible assembly of homogenous high-order aggregates and, thus, the intensity of immunogenic response cannot be accurately correlated with such molecules. Consequently, FDA guidelines currently recommend that that the presence of aggregates in biotherapeutic drug products are minimised, not only at manufacture but for the duration of the product's shelf-life.
The primary experimental strategy for the production of a highly pure biotherapeutic product centres on the physical separation, or otherwise known as fractionation, of the protein monomer from its aggregated counterparts within a mixture. Fractionation is principally achieved through liquid chromatography, a technique that uses a liquid flow through specifically functionalised columns to influence the elution of protein molecules by exploiting a range of molecular properties, such as size, binding affinity and electrostatic charge. Since the monomer and aggregates do not share the same molecular properties, their elution from functionalised columns is temporally differentiated, and isolation of the purified monomer is possible. Clearly, the analytical power in this technique is only truly realised when it is coupled with a detector that can monitor and visualise the fractionated species eluting over time. Examples of various techniques that are either currently used, or could be used, as liquid chromatography detectors are summarised in Table 1 on the following page.
TABLE 1A summary of properties of various detectors coupled with existing liquidchromatography systems.Used tocontrolfractionDetectorPropertyInformationAdvantageDisadvantagecollectorUltraviolet (UV)Absorbance ofConcentrationInexpensive; widelyNo identity information;Ylight at a specificif detector isapplicable for proteindoesn't respond towavelengthcalibrated &analysisstructural changesRefractive IndexRefraction of lightextinctionInexpensiveSignal instability with bufferY(RI)compared tocoefficientchanges or environmentalreferenceknowncondition e.g. salt gradientsand temperatureFluorescenceEmission of lightInexpensive; sensitiveRequires fluorescent probe;Y1at a specificsmall linear range due towavelengthsensitivityMassMass to chargeMolecularIdentification of elutingExpensive; destructive; notY2Spectrometryratiomassspecies positivelycompatible with excipients;(MS)confirmedrequires compatible buffer;cannot work at formulationconcentrations; ionisationcan disrupt weaklyassociated; automateddetection of intact proteinsnot availableNuclear MagneticProbing3D structureDetailed structuralExpensive; Large footprint;NResonanceelectromagneticinformationLong analysis times; Labour(NMR)field of nucleiintensive; Requireswith NMR-activeexperienced person for dataspininterpretation; non-continuous detection;mainly used for smallmolecules; requireddeuterated solvents andsample modification; notcompatible with formulationexcipientsElementalAbsorbance ofConcentrationSpecific way to followDestructive; Usually used inNanalysislight at discreteof elementelution of targetconjunction with(AAS/ICP-OES)wavelengths byunder studyprotein if metalconcentration detector;free atoms in abindingunless metal stoichiometrygaseous stateknown then protein speciescannot be identifiedEvaporative lightLight scattered byMassRelatively inexpensiveNot applicable for proteinYscatteringmolecule in gasapplications as techniquephaserequires gas phaseNotes:1See for example U.S. Pat. No. 5,541,4202See R. Moritz and N. O'Reilly, J Biomol Tech. 2003 June; 14(2): 136-142
Ultraviolet (UV) detectors are common amongst detectors coupled with liquid chromatography systems for the purpose of profiling protein mixtures for isolation or purification of their components. This is largely due to their cost-effectiveness and wide applicability for protein samples. UV detectors capitalise on a natural and quantifiable phenomenon common to all proteins arising from chromophores: functional groups that are able to absorb light at specific wavelengths. In proteins, for example, the peptide bond absorbs light at 215 nm and the aromatic group on certain amino acids absorbs at 280 nm. No modification is therefore required to be made to the proteins to determine their presence under UV light, and analyses can occur with the protein unmodified.
Signal responses from refractive index (RI) and fluorescence detectors are also quantifiable, but their use in protein purification applications is limited due to signal instability with changes in buffer components and the need for a fluorescent probe, respectively. Concentration information is inherent in the data from all three types of detectors, but no further information confirming the identity of the fractionated species can be extracted from such measurements. Such confirmation would require the implementation of additional offline methods, which contribute significantly to the total analysis and handling times and, correspondingly, decrease efficiency and profitability.
To provide identity information, mass spectrometry (MS) and nuclear magnetic resonance (NMR) technologies in tandem with liquid chromatography systems may be used. However, the additional information obtained through these techniques comes at a price, including increased equipment and running costs and prolonged analysis times. MS and NMR also suffer from sample-specific issues, where the excipients used in biopharmaceutical formulations are frequently incompatible with either technique, as are the concentrations of the protein within. Whether MS accurately reflects the species contained within the original solution is another critical issue for consideration in biopharmaceutical analyses. Since this technique relies on the ionisation of charged groups, the likelihood of observing a given species is directly correlated with the accessibility of these charged sites—which may or may not be the same for two aggregate particles of the same mass—and, furthermore, the ionisation itself may also cause dissociation of weakly associated molecules.
Following successful fractionation of a biopharmaceutical or protein/aggregate mixture, the next consideration is isolation of the fractionated protein monomer. UV, RI, fluorescence and mass spectrometer technologies that facilitate the isolation of fractionated species by controlling a fraction collector are commercially available. This signal-based fraction collection is largely accomplished through user-set parameters that define specific conditions during which a particular fraction should be collected. Such conditions may for example relate to changes in peak slope or absolute signal above a threshold value. The disadvantage of using UV- or RI-directed fraction collection for biopharmaceutical mixtures is that these detectors lack the ability to discriminate between a protein monomer and its aggregates. For example, there is no discernable difference in the signal response for 10 individual monomers or an aggregate composed of 10 monomers. This means that the efficiency of the peak fractionation, i.e. the ability to identify where peaks begin and end in both baseline resolved and unresolved situations, is solely based on the ability to detect changes in the intensity of the resultant peaks during real-time measurements of the phenomenon under observation.
More selective fraction collection based on species identity is possible with mass spectrometry, where a given fraction is collected based on the detection of a particular mass. However, it is important to remember that when looking at intact proteins that the observed species are presented as a mass to charge ratio; therefore, as far as the MS detector is concerned a species of mass 100 and charge of +10 would be the same as a species of mass 10 and charge of +1. No real-time deconvolution of such data is currently possible. Even when looking at peptide fingerprints, such selective fractionation excludes all information from other molecules and as a result does not allow the user to effectively minimise contamination of the fraction from other closely eluting species. Similar drawbacks are applicable for selective fractionation based on fluorescence or elemental analyses.
Ensuring a high purity product at maximum yield is a specific manufacturing challenge faced by the biopharmaceutical industry, on both small and large scales, which is required to meet stringent regulatory demands whilst maintaining profitability. Concomitantly, the demand for technologies that detect, characterise and quantify aggregates, both during and post-manufacture, has increased and manufacturers of scientific instrumentation have duly responded. The benefits of using detectors for online monitoring in polymer manufacture have been recognised and implemented with the optimisation tool ACOMP (automatic continuous online monitoring of polymerisation reaction, as described for example in U.S. Pat. Nos. 6,052,184 and 6,653,150). This tool aims to increase efficient production of a polymerisation reaction by adjusting variables, such as pressure, temperature and reagent concentrations, based on the feedback from real-time analyses of several parameters. However, no technology currently available offers real-time, signal-based fraction collection or identity-based selective fractionation. All existing technologies for analysis of protein/aggregate mixtures require some degree of user intervention for data processing, and none currently allow for automated, software-driven identification of monomer and aggregate peaks or corresponding fractions.
It is an object of the invention to address one or more of the above mentioned problems.